High-Throughput Sequencing technologies are providing unprecedented inventories of microbial communities in aquatic samples, offering an invaluable tool to estimate the impact of anthropogenic pressure on marine communities. In this case study, the Mediterranean touristic site of Aci Castello (Italy) was investigated by High-Throughput Sequencing of 16S and 18S rRNA genes. The sampling area falls within a Marine Protected Area and, notwithstanding, features an untreated urban wastewater discharge. Seawater samples were collected close to the wastewater output (COL) and at a second station about 400 m further off (PAN), before and after a summer increase in population. Prokaryotic communities clustered according to stations, rather than to seasons. While PAN showed a typical, not impacted, marine microbial composition, COL was consistently enriched in Epsilonproteobacteria and Firmicutes …